Hyaluronan is a high molecular weight glycosaminoglycan that is associated with a number of normal and pathologic biological processes involving cellular migration and/or inflammation including: embryogenesis; fetal development; tumorigenesis; and wound repair. This laboratory has recently demonstrated differential phenotypic and cell specific expression of the three different hyaluronan synthases (HAS) transcripts in mesenchymal cells treated with proinflammatory cytokines. The objectives of this proposal are to determine the mechanisms involved in proinflammatory cytokine regulation of hyaluronan expression and to determine the role of these proinflammatory cytokines in regulating the in vivo expression of hyaluronan during wound repair. The information generated by this study is relevant not only to wound repair, but also to a spectrum of other inflammatory processes including rheumatoid arthritis, inflammatory bowel diseases, and tumorigenesis. To fulfill these objectives the following specific aims will be examined: 1. To identify and characterize the interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-alpha) response elements in the HAS promoters. The promoters for the human HAS genes will be isolated. IL-1 and TNF-alpha responsive cis-and trans-acting elements will be identified using electrophoretic mobility shift assays, super shift assays, and nuclease footprint assays. Antisense oligonucleotides will be used to directly test whether identified transcription factors are involved in proinflammatory induced HAS regulation. 2. To identify the signaling transduction pathway(s) involved in the proinflammatory cytokine induction of the HAS promoters. A battery of inhibitors, kinase- specific antisera, and promoter/reporter constructs will be used to identify the signaling transduction pathways involved in IL-1 and TNF-alpha induction of HAS gene expression. 3. To determine the HAS protein(s) responsible for synthesizing hyaluronan in response to proinflammatory cytokine induction. Levels of synthesis, molecular weight ranges, and localization of hyaluronan generated from full-length cDNAs will be determined. HAS-specific antisera and antisense oligonucleotides will be used to examine the expression of individual enzymes. Rates of hyaluronan synthesis and enzyme stabilities and kinetics will be established. 4. To establish the role of the proinflammatory cytokines, IL-1 and TNF-alpha in regulating the in vivo expression of hyaluronan. A longitudinal analysis of hyaluronan expression will be performed using a murine model. Mice containing knockout lesions in 1) the IL-1 receptor, 2) both of the TNF receptors, and 3) a combination IL-1 and TNF-alpha receptor knockouts will be used to determine the role of IL-1 and TNF-alpha in regulating hyaluronan expression during repair.